This is a competitive renewal of NIH grant DK55545 which is focused on the role of PI 3-kinase in insulin action. PI 3-kinase is important as a critical node in insulin control of metabolism and a key point of divergence of insulin signaling. Using, both in vitro and in vivo approaches, including RNA interference (RNAi) and creation and characterization of mice and cell lines in which specific isoforms of PI 3-kinase have been deleted, i.e. knocked out, at the whole body or tissue specific levels, we have demonstrated multiple, important ways in which this enzyme controls insulin signaling in both positive and negative ways. This includes differences in the activity and properties of the various regulatory subunits of PI 3-kinase, the important role of stoichiometry between regulatory and catalytic subunits in PI 3-kinase signaling and insulin action, and the ability of PI 3-kinase to allow divergence of the downstream signal between Akt and atypical PKCs. In addition, we have demonstrated alterations in PI 3-kinase activity in disease states and the relationship of this pathway to other signaling pathways, including two previously unrecognized connections: one between PI 3-kinase regulatory subunits and activation of the stress kinases JNK and p38, which may link the p85 subunit to serine phosphorylation of IRS proteins in insulin resistant states;and a second between the PI 3-kinase regulatory subunit and the activity of the major PIPS phosphatase PTEN in cells. This has led us to new hypotheses about the important role of PI 3-kinase not only as a site of divergence of the insulin signaling pathways, but also a site of both positive and negative regulation in physiological and pathological states, and a site for cross-talk with other signaling systems, especially the stress kinases. In the next five years, we propose to 1) Dissect the multiple, differential roles of PI 3-kinase regulatory subunits (p85a/b, p50a and p55a/AS53) in insulin signaling, focusing on the potential actions and interactions emanating from the different N-terminal domains of the regulatory subunits that are independent of PI 3- kinase activity;2) Determine the role of the PI 3-kinase catalytic subunits p110a and p11 Ob in divergent insulin signaling in vivo through tissue specific deletion;and 3) Explore new chemical biology approaches to modifying PI 3-kinase mediated signaling in normal and insulin resistant states through the use of small molecule screening and new in vivo gene silencing techniques using nanoparticle delivery systems.